High performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate

Abstract

The invention discloses a high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate, and belongs to the technical field of pharmaceutical analysis. According to the method, a chromatographic column taking octadecyl bonded silica gel as a filling agent is adopted, an ammonia water solution is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, gradient elution is carried out, and genotoxic impurities in a chlorpheniramine maleate sample are detected by utilizing a high performance liquid chromatography; and the genotoxic impurities are 2-aminopyridine-N-oxide, 2-bromopyridine-N-oxide and 2-aminopyridine. The invention further discloses a preparation method of the genotoxic impurities. The method disclosed by the invention can be used for simultaneously detecting three trace genotoxic impurities, is high in sensitivity, good in separation degree and low in product matrix interference, can be widely applied to a high performance liquid chromatography detection method for determining the trace genotoxic impurities in the chlorpheniramine maleate, fully meets the qualitative and quantitative analysis of the trace genotoxic impurities in the product, and has a wide application prospect. And the product quality is effectively controlled.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a high performance liquid chromatography detection method for trace genotoxic impurities in chlorphenamine maleate. Background Art

[0002] Chlorphenamine maleate, with the INN name of Chlorphenamine maleate, has the chemical name of 2-[p-chloro-α-[2-(dimethylamino)ethyl]phenyl]pyridine maleate, molecular formula: C 16 H 19 ClN2·C4H4O4, molecular weight: 390.86, and the chemical structural formula is:

[0003]

[0004] Chlorphenamine maleate is an active pharmaceutical ingredient produced and sold by our company. As a first-generation H1 receptor antagonist, it can selectively block histamine receptors, antagonize the effects of histamine and produce antihistamine effects; it can counteract the capillary dilation caused by allergic reactions (histamine), reduce the permeability of capillaries, and relieve the wheezing caused by bronchial smooth muscle contraction. The antihistamine effect of this product is relatively persistent, and it also has obvious central inhibitory effects, which can enhance the effects of anesthetics, analgesics, hypnotics and local anesthetics. Its main indications are skin allergic diseases such as urticaria, eczema, dermatitis, drug rash, pruritus, neurodermatitis, insect bite dermatitis, solar dermatitis, and it can also be used for allergic rhinitis, drug and food allergies. Clinically, it is used for various allergic diseases, and can also be combined with other Chinese and Western medicines as one of the main components of compound anti-cold medicines.

[0005] The applicant synthesizes chlorphenamine maleate using p-chloroacetonitrile, 2-bromopyridine, dimethylaminoethyl chloride hydrochloride and maleic acid as starting materials. Through substitution, condensation, hydrolysis, the synthesis of crude chlorphenamine maleate, and the refinement of the finished product of chlorphenamine maleate, a total of 5 processes are carried out to obtain chlorphenamine maleate. The synthetic process route is as Figure 1 shown.

[0006] According to Figure 1 the synthetic route, it can be seen that the applicant uses the starting materials dimethylaminoethyl chloride hydrochloride and 2-bromopyridine in the production process. Among them, the theoretically degraded products and process by-products of 2-bromopyridine, namely GTI-1 (2-aminopyridine-N-oxide), GTI-2 (2-bromopyridine-N-oxide), and GTI-3 (2-aminopyridine), are all potential genotoxic impurities, and their structural formulas are shown in Table 1 below.

[0007] Table 1 List of Potential Genotoxic Impurities

[0008]

[0009] Genotoxic impurities (GTIs) refer to chemical substances that can directly or indirectly damage DNA, cause gene mutations or chromosomal aberrations, and may trigger cancer. The control of them is one of the core challenges in drug safety. According to the ICH M7 guideline, genotoxic impurities can be classified into five categories, namely, Category 1: known to be mutagenic and carcinogenic (such as nitrosamine compounds); Category 2: known to be mutagenic but unknown carcinogenicity; Category 3: containing warning structures (such as aromatic amines, alkyl sulfonates) but without mutagenicity data; Category 4: warning structures are the same as those of the active pharmaceutical ingredient and without mutagenicity; Category 5: without warning structures or proven to be risk-free. According to the above classification, GTI-1, GTI-2, and GTI-3 that may be contained in our chlorpheniramine maleate belong to Category 3. According to the ICH M7 guideline, calculate the limits of the above potential genotoxic impurities in chlorpheniramine maleate based on a safe dose of 1.5 μg / day. Combining with the maximum daily dose of chlorpheniramine maleate being 20 mg / day, the calculation results are as follows:

[0010]

[0011] From the above calculation results, it can be seen that the possible amounts of GTI-1, GTI-2, and GTI-3 contained in chlorpheniramine maleate are very low and cannot exceed 75 ppm. Therefore, the detection methods for these three genotoxic impurities are required to have high sensitivity, good resolution, and low product matrix interference to meet the stringent requirements for the detection of trace genotoxic impurities. At present, there is no report on a method for simultaneously detecting three trace genotoxic impurities GTI-1, GTI-2, and GTI-3 in chlorpheniramine maleate. Therefore, there is an urgent need to provide a detection method with high sensitivity, good resolution, low product matrix interference, and rapidity to provide technical support for the quality control of chlorpheniramine maleate and its preparations. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a high-performance liquid chromatography detection method that can simultaneously detect three trace genotoxic impurities, has high sensitivity, good resolution, and low product matrix interference, and can be widely used for the determination of trace genotoxic impurities in chlorpheniramine maleate, fully meeting the qualitative and quantitative analysis of trace genotoxic impurities in the product and effectively controlling the product quality.

[0013] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0014] A high performance liquid chromatography (HPLC) method for detecting trace genotoxic impurities in chlorpheniramine maleate. A chromatographic column filled with octadecylsilyl silica gel is used. Ammonia aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution. The genotoxic impurities in the chlorpheniramine maleate sample are detected by HPLC. The genotoxic impurities are 2-aminopyridine-N-oxide, 2-bromopyridine-N-oxide, and 2-aminopyridine.

[0015] Preferably, the concentration of the ammonia aqueous solution is 1 mL / L.

[0016] Preferably, the chromatographic column is a Welch Xtimate C18 chromatographic column with a specification of 4.6 mm × 150 mm, a packing particle size of 5 μm, and a column temperature of 30 °C.

[0017] Preferably, the gradient elution program is as follows:

[0018] Time (min) Mobile Phase A / % Mobile Phase B / % 0 97 3 6 97 3 11 90 10 12 5 95 19 5 95 19.5 97 3 25 97 3

[0019] The above percentages are by volume.

[0020] Preferably, the flow rate of the gradient elution is 1 mL / min.

[0021] Preferably, in the HPLC method, the injection volume is 5 μL.

[0022] Preferably, in the HPLC method, the detection wavelength is 225 nm.

[0023] Preferably, the steps for detecting the genotoxic impurities in the chlorpheniramine maleate sample include the following:

[0024] 1) Prepare a test solution and a reference solution containing genotoxic impurities.

[0025] 2) Detect the test solution and the reference solution by HPLC, and record the peak area of the corresponding genotoxic impurity in the test solution, denoted as A 供试品 , and the peak area of the genotoxic impurity in the reference solution, denoted as A 对照品 ;

[0026] 3) Calculate the content ppm of the genotoxic impurity in chlorpheniramine maleate according to the following formula:

[0027]

[0028] In the formula:

[0029] W 对照品 is the weighed amount of the reference substance, mg;

[0030] A 对照品is the peak area of the genotoxic impurity in the reference solution;

[0031] V 对照品 is the dilution factor of the reference substance;

[0032] W 供试品 is the weighed amount of the test sample, mg;

[0033] A 供试品 is the peak area of the genotoxic impurity in the test sample solution;

[0034] V 供试品 is the dilution factor of the test sample.

[0035] Preferably, the preparation of the reference solution containing genotoxic impurities includes the following steps:

[0036] S1. Respectively take 23 mg of 2-aminopyridine-N-oxide, 2-bromopyridine-N-oxide and 2-aminopyridine, place them in 25-mL volumetric flasks respectively, dissolve with acetonitrile and dilute to the scale, shake well, and prepare 2-aminopyridine-N-oxide stock solution, 2-bromopyridine-N-oxide stock solution, and 2-aminopyridine stock solution respectively;

[0037] S2. Respectively pipette 1 mL of 2-aminopyridine-N-oxide stock solution, 2-bromopyridine-N-oxide stock solution and 2-aminopyridine stock solution into a 25-mL volumetric flask, dilute to the scale with the solvent, and shake well to prepare the reference stock solution;

[0038] S3. Pipette 1 mL of the reference stock solution into a 25-mL volumetric flask, dilute to the scale with the solvent, and shake well to prepare the reference solution.

[0039] Preferably, the preparation process of the test sample solution is as follows: Take 20 mg of the test sample, weigh accurately, place it in a 2-mL EP tube, dissolve with 1 mL of the solvent, and shake well. The test sample solution is prepared.

[0040] Preferably, the solvent is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:9. Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0041] 1) Compared with the prior art, the present invention uses 0.1% ammonia aqueous solution as the mobile phase. By using the alkaline mobile phase, the elution time is shorter, and the chromatographic column used is alkali-resistant;

[0042] 2) The detection method of the present invention can simultaneously and rapidly detect three trace genotoxic impurities GTI-1, GTI-2, and GTI-3 in chlorpheniramine maleate. It has good specificity, sensitivity, accuracy, etc., low product matrix interference, and simple operation, fully meeting the qualitative and quantitative analysis of trace genotoxic impurities in the product, and effectively controlling the product quality. Description of the Drawings

[0043] Figure 1 This is the synthetic route diagram of the background art of the present invention;

[0044] Figure 2 This is the chromatogram of the control solution detected in Example 1 of the present invention;

[0045] Figure 3 This is the chromatogram of the test solution detected in Example 1 of the present invention;

[0046] Figure 4 This is the chromatogram of the spiked test solution detected in Example 1 of the present invention;

[0047] Figure 5 This is the chromatogram of Comparative Example 11;

[0048] Figure 6 This is the chromatogram of Comparative Example 12. Detailed Description of the Invention

[0049] The following further illustrates the present invention in conjunction with specific embodiments. The embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0050] Example 1

[0051] (1) Detection conditions

[0052] Instrument: High performance liquid chromatograph Agilent1200;

[0053] Detection wavelength: 225 nm;

[0054] Chromatographic column: Welch Xtimate C18, with a specification of 4.6 mm × 150 mm and a packing particle size of 5 μm;

[0055] Mobile phase A: 0.1% ammonia aqueous solution (add 1.0 mL of ammonia water to 1 L of ultrapure water and sonicate for 10 min);

[0056] Mobile phase B: Acetonitrile;

[0057] The gradient elution program is shown in Table 2:

[0058] Table 2 Gradient elution program

[0059] Time (min) Mobile Phase A / % Mobile Phase B / % 0 97 3 6 97 3 11 90 10 12 5 95 19 5 95 19.5 97 3 25 97 3

[0060] Flow rate: 1 mL / min;

[0061] Column temperature: 30 °C;

[0062] Sample injection volume: 5 μL;

[0063] Solvent: Acetonitrile: water = 1:9;

[0064] (2) Preparation of sample solutions

[0065] Test solution: Take 20 mg of the test substance, accurately weigh it, place it in a 2 mL EP tube, add 1.0 mL of the solvent to dissolve it, and shake well;

[0066] GTI-1 stock solution 1: Take 23 mg of impurity GTI-1, accurately weigh it, place it in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well;

[0067] GTI-2 stock solution 1: Take 23 mg of impurity GTI-2, accurately weigh it, place it in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well;

[0068] GTI-3 stock solution 1: Take 23 mg of impurity GTI-3, accurately weigh it, place it in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well;

[0069] Reference stock solution 2: Pipette 1.0 mL each of GTI-1 stock solution 1, GTI-2 stock solution 1, and GTI-3 stock solution 1 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well;

[0070] Reference solution: Pipette 1.0 mL of reference stock solution 2 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well.

[0071] Fortified test solution: Take 20 mg of the test substance, accurately weigh it, place it in a 2 mL EP tube, add 1.0 mL of the reference solution to dissolve it, and shake well.

[0072] Blank solution: Acetonitrile: water = 1:9 (V:V).

[0073] (3) Injection and detection

[0074] Inject the above blank solution, reference solution, and test solution respectively. The specific injection sequence is shown in Table 3 below. The injection sequence should use a concomitant reference (reference solution). Take the first 5 injections of the reference solution and 1 injection of the concomitant reference, calculate the RSD of the peak areas, and the requirement is RSD ≤ 15.0%; if multiple concomitant references are injected, calculate in the same way, and the requirement is RSD ≤ 15.0%.

[0075] Table 3 Injection sequence

[0076]

[0077]

[0078] The blank solution, reference solution, test solution, and spiked test solution were detected according to the instruments and chromatographic conditions disclosed in step (1), and the chromatograms were recorded. The typical chromatograms are as shown in Figures 2 - 4 shown.

[0079] As can be seen from Figure 2 , under the chromatographic conditions of the present invention, GTI-1 (2-aminopyridine-N-oxide), GTI-2 (2-bromopyridine-N-oxide), and GTI-3 (2-aminopyridine) have strong retention ability, with good peak shapes and good resolution, meeting the quantitative requirements.

[0080] As can be seen from Figure 3 , other impurities contained in chlorpheniramine maleate in the test solution do not peak at the elution positions of GTI-1, GTI-2, and GTI-3, without matrix effect and will not interfere with the detection of GTI-1, GTI-2, and GTI-3.

[0081] As can be seen from Figure 4 , the main peak of chlorpheniramine maleate and the solvent peak do not interfere with the elution of GTI-1, GTI-2, and GTI-3, and have good resolution, without affecting the quantification of GTI-1, GTI-2, and GTI-3.

[0082] Example 2

[0083] 1. System suitability

[0084] GTI-1 stock solution 1: Take 23 mg of impurity GTI-1, accurately weigh, place in a 25 mL volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well to obtain;

[0085] GTI-2 stock solution 1: Take 23 mg of impurity GTI-2, accurately weigh, place in a 25 mL volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well to obtain;

[0086] GTI-3 stock solution 1: Take 23 mg of impurity GTI-3, accurately weigh, place in a 25 mL volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well to obtain;

[0087] Reference stock solution 2: Pipette 1.0 mL each of GTI-1 stock solution 1, GTI-2 stock solution 1, and GTI-3 stock solution 1 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0088] Reference solution: Pipette 1.0 mL of reference stock solution 2 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0089] Blank solution: acetonitrile: water = 1:9 (V:V).

[0090] The chromatographic conditions of Example 1 were followed, and after the system was balanced, 2 injections of blank solution and 6 injections of reference solution were taken, and the chromatogram was recorded. The separation degree of the analyte peak and the adjacent chromatographic peak in the first injection of the reference solution was recorded, and the peak area, peak area mean and RSD value of the analyte injected 6 times in a row were recorded. The results are shown in Tables 4 and 5.

[0091] Table 4 System suitability results

[0092]

[0093]

[0094] Table 5 Intermediate precision results

[0095]

[0096]

[0097] It can be seen from Tables 4 and 5 that the minimum separation between the analyte and the adjacent chromatographic peak in the first injection of the reference solution is 5.0 (≥1.5); the maximum RSD of the peak area of ​​the analyte after 6 consecutive injections of the reference solution is 2.09% (RSD≤15.0%); the maximum RSD of the peak area of ​​the analyte in the first 5 injections of the reference solution and the accompanying control is 1.37% (RSD≤15.0%), and the system applicability of the method of the present invention meets the detection requirements.

[0098] 2. Exclusivity

[0099] GTI-1 stock solution 1: Take 23 mg of impurity GTI-1, accurately weigh it, put it in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well to obtain;

[0100] GTI-2 stock solution 1: Take 23 mg of impurity GTI-2, accurately weigh it, put it in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well to obtain;

[0101] GTI-3 stock solution 1: Take 23 mg of impurity GTI-3, weigh accurately, place in a 25 mL volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well to obtain;

[0102] GTI-1 peak attribution solution: Pipette 1.0 mL of GTI-1 stock solution 1 into a 50 mL volumetric flask, dilute to scale with solvent, and shake well to obtain;

[0103] GTI-2 peak attribution solution: Pipette 1.0 mL of GTI-2 stock solution 1 into a 50 mL volumetric flask, dilute to scale with solvent, and shake well to obtain;

[0104] GTI-3 Peak Attribution Solution: Pipette 1.0 mL of GTI-3 Stock Solution 1 into a 50 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0105] Reference Standard Stock Solution 2: Pipette 1.0 mL each of GTI-1 Stock Solution 1, GTI-2 Stock Solution 1, and GTI-3 Stock Solution 1 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0106] Reference Standard Solution: Pipette 1.0 mL of Reference Standard Stock Solution 2 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0107] Test Solution: Take 20 mg of the test sample, accurately weigh it, transfer it to a 2 mL EP tube, add 1.0 mL of the solvent to dissolve it, and shake well to obtain.

[0108] Fortified Test Solution: Take 20 mg of the test sample, accurately weigh it, transfer it to a 2 mL EP tube, add 1.0 mL of the reference standard solution to dissolve it, and shake well to obtain;

[0109] Blank Solution: Acetonitrile: Water = 1:9 (V:V).

[0110] Operate according to the chromatographic conditions in Example 1 above. After the system suitability is qualified, take the blank solution, reference standard solution, test solution, and fortified test solution, inject 1 injection each, and record the chromatogram. Record the retention time, peak area of each analyte in each solution, and the resolution from the adjacent chromatographic peaks. The results are shown in Table 6.

[0111] Table 6 Results of Specificity Test

[0112]

[0113]

[0114] As can be seen from Table 6, compared with the chromatogram of the blank solution and the reference standard solution, there is no interference in the blank solution at the elution position of the analyte; the minimum resolution of the analyte from the adjacent chromatographic peaks in the reference standard solution, test solution, and fortified test solution is 2.8 (≥1.5); the retention times of the analyte peaks in the reference standard solution, test solution, and fortified test solution are consistent; the analyte peak in the fortified test solution is enhanced compared with the corresponding peak in the test solution, and the method of the present invention has good specificity.

[0115] 3. Detection Limit and Quantification Limit

[0116] Detection Limit Solution: Pipette 1.0 mL of the reference standard solution prepared in "1. System Suitability" into a 10 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0117] Quantitation limit solution: Pipette 3.0 mL of the reference solution into a 10 mL volumetric flask, dilute to the mark with the solvent, and mix well to obtain the solution.

[0118] After the system suitability is qualified, operate according to the chromatographic conditions in Example 1 above. Inject 3 needles each of the quantitation limit solution and the detection limit solution, and record the chromatograms. Record the peak area, RSD of the peak area, and signal-to-noise ratio (S / N) of the analyte in the quantitation limit solution; the peak area and signal-to-noise ratio (S / N) of the analyte in the detection limit solution; calculate the concentrations of the quantitation limit solution and the detection limit solution and the percentage of this concentration equivalent to the concentration of the test solution. The results are shown in Table 7.

[0119] Table 7 Results of Detection Limit and Quantitation Limit

[0120]

[0121]

[0122] As can be seen from Table 7, the maximum RSD of the peak area of the analyte in the quantitation limit solution for 3 consecutive injections is 3.68% (≤15.0%); the minimum signal-to-noise ratio of the analyte in the quantitation limit solution is 25.6 (≥10). The quantitation limit of this method for GTI-1 is 0.4402 μg / mL (equivalent to 22.01 ppm of the concentration of the test solution), for GTI-2 is 0.4365 μg / mL (equivalent to 21.82 ppm of the concentration of the test solution), and for GTI-3 is 0.4310 μg / mL (equivalent to 21.55 ppm of the concentration of the test solution). The quantitation limit meets the detection requirements.

[0123] 4. Repeatability

[0124] Reference solution: Pipette 1.0 mL of the reference stock solution 2 prepared in "1. System Suitability" into a 25 mL volumetric flask, dilute to the mark with the solvent, and mix well to obtain the solution;

[0125] Repeatability solution: Take 20 mg of the test sample, accurately weigh it, place it in a 2 mL EP tube, add 1.0 mL of the reference solution to dissolve it, mix well to obtain the solution, and prepare 6 parallel portions.

[0126] After the system suitability is qualified, operate according to the chromatographic conditions in Example 1 above. Take 6 portions of the repeatability solution, inject 1 needle each, and calculate the content of the analyte in the 6 portions of the repeatability solution based on the average value of the peak area of the reference solution in the system suitability. Record the peak area, individual content value, Mean±SD, RSD value, and 95% confidence interval of the analyte. The results are shown in Table 8.

[0127] Table 8 Results of Repeatability Test

[0128]

[0129]

[0130] As can be seen from Table 7, the maximum RSD of the analyte content in 6 replicates of the solution was 1.69% (≤15.0%).

[0131] 5. Linearity and range

[0132] Reference stock solution 2: Pipette 1.0 mL each of GTI-1 stock solution 1, GTI-2 stock solution 1, and GTI-3 stock solution 1 into a 25-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0133] Quantitation limit solution: Pipette 3.0 mL of the reference solution prepared in "1. System suitability" into a 10-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0134] Reference solution: Pipette 1.0 mL of reference stock solution 2 into a 25-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0135] 50% linear solution: Pipette 0.5 mL of reference stock solution 2 into a 25-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0136] 75% linear solution: Pipette 0.75 mL of reference stock solution 2 into a 25-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0137] 150% linear solution: Pipette 1.5 mL of reference stock solution 2 into a 25-mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain.

[0138] After the system suitability is qualified, operate according to the chromatographic conditions of Example 1 above. Inject 1 injection each of the quantitation limit solution and each linear solution, and record the chromatogram. Record the actual concentration and peak area of each linear solution, perform a univariate linear regression of the peak area against its concentration, calculate the linear correlation coefficient r, y-axis intercept, and sum of squared residuals, 95% confidence interval of the y-axis intercept, and absolute value of the peak area corresponding to the y-axis intercept / 100% concentration. The results are shown in Table 9.

[0139] Table 9 Results of linearity test

[0140]

[0141]

[0142] As can be seen from Table 9, the minimum value of the linear correlation coefficient r of the analyte is 0.9984 (≥0.990); the maximum value of the ratio of the absolute value of the Y-axis intercept to the peak area corresponding to 100% concentration is 5.8% (≤25.0%). The method of the present invention shows a linear relationship in the range of 0.4402 μg / mL to 2.2009 μg / mL (LOQ to 150% concentration level) for GTI-1; a linear relationship in the range of 0.4365 μg / mL to 2.1825 μg / mL (LOQ to 150% concentration level) for GTI-2; and a linear relationship in the range of 0.4310 μg / mL to 2.1551 μg / mL (LOQ to 150% concentration level) for GTI-3.

[0143] 6. Accuracy

[0144] Test solution: Take 20 mg of the test sample, weigh accurately, place it in a 2 mL EP tube, add 1.0 mL of solvent to dissolve, and shake well to obtain;

[0145] Quantitation limit solution: Pipette 3.0 mL of the reference solution prepared in "1. System suitability" into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain;

[0146] Reference solution: Pipette 1.0 mL of the reference stock solution 2 prepared in "1. System suitability" into a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain;

[0147] 150% linear solution: Pipette 1.5 mL of the reference stock solution 2 into a 25 mL volumetric flask, dilute to the mark with solvent, and shake well to obtain;

[0148] Accuracy solution: Take 20 mg of the test sample, weigh accurately, place it in a 2 mL EP tube, and prepare 9 portions in parallel; dissolve by adding 1.0 mL of different concentration solutions (3 portions for each concentration level) according to Table 10 below, shake well to obtain a total of 9 accuracy solutions.

[0149] Table 10 Preparation of accuracy solutions

[0150] Spiking Level 30% (LOQ) Spiked Solution 100% Spiked Solution 150% Spiked Solution Added Solution Quantification Limit Solution Reference Solution 150% Linear Solution Added Volume (mL) 1.0 1.0 1.0

[0151] After the system suitability is qualified, operate according to the chromatographic conditions of Example 1 above. Inject 1 injection each of the test solution and 9 accuracy solutions, and record the chromatograms. Calculate and report the content of the analyte in the test solution before and after spiking, the measured added amount, the theoretical added amount, the single value of the recovery rate, the average value of the recovery rate, the RSD value, and the 95% confidence interval. The results are shown in Table 11.

[0152] Table 11 Results of recovery test

[0153]

[0154]

[0155]

[0156] As can be seen from Table 11, the recovery rate ranges of the 9 accuracy solutions of GTI-1 are from 95.5% to 97.6%; those of GTI-2 are from 96.7% to 99.8%; those of GTI-3 are from 95.6% to 112.3% (between 70.0% and 130.0%); the maximum RSD of the overall recovery rate (n = 9) is 6.30% (≤15.0%), indicating that the accuracy of the method of the present invention is good.

[0157] In summary, the detection method of the present invention can simultaneously and rapidly detect three trace genotoxic impurities GTI-1, GTI-2, and GTI-3 in chlorpheniramine maleate, with good specificity, sensitivity, accuracy, etc., low matrix interference of the product, simple operation, fully meeting the qualitative and quantitative analysis of trace genotoxic impurities in the product, and effectively controlling the product quality.

[0158] Example 3

[0159] In Comparative Examples 1 to 8, except for the following conditions, other parameters and operations were the same as those in Example 1, and the specific test conditions and test results are shown in Table 12.

[0160] Reference solution: Pipette 1.0 mL of reference stock solution 2 into a 25 mL volumetric flask, dilute to the mark with the solvent, and shake well to obtain;

[0161] Test sample spiked solution: Take 20 mg of the test sample, accurately weigh it, place it in a 2 mL EP tube, add 1.0 mL of the reference solution to dissolve it, and shake well to obtain.

[0162] 1. Operate according to the chromatographic conditions in Comparative Examples 1 to 8, inject the reference solution and the test sample spiked solution, record the chromatogram, and calculate the content of each genotoxic impurity. The results are shown in Table 12.

[0163] Table 12 Test Results of Comparative Examples 1 to 8

[0164]

[0165] 2. Operate according to the chromatographic conditions in Comparative Examples 9 to 10, inject the reference solution and the test sample spiked solution, record the chromatogram, and calculate the content of each genotoxic impurity. The results are shown in Tables 13 to 14.

[0166] Table 13 Gradient Elution Program

[0167]

[0168]

[0169] Table 14 Test Results of Comparative Examples 9 - 10

[0170]

[0171] From the detection results of the above Comparative Examples 1 - 10, it can be seen that under the chromatographic conditions of Example 1 of the present invention, by finely adjusting chromatographic parameters such as wavelength, column temperature, flow rate, alkali concentration, and elution gradient, and using the finely adjusted detection conditions to detect the contents of three trace genotoxic impurities in chlorpheniramine maleate, they can all be accurately detected. Therefore, the established detection method of the present invention has good durability.

[0172] Comparative Example 11

[0173] According to the method and chromatographic conditions of chlorpheniramine maleate injection in the Chinese Pharmacopoeia, the specific detection steps are as follows:

[0174] (1) Detection Conditions

[0175] Detection wavelength: 262 nm;

[0176] Chromatographic column: Octadecylsilyl bonded silica gel chromatographic column (Agilent ZORBAX SB C8), with a specification of 4.6 * 150 mm and a packing particle size of 5 μm;

[0177] Isocratic elution, mobile phase: Acetonitrile - aqueous solution containing 5% phosphoric acid and 5% triethylamine (20:80);

[0178] Flow rate: 1 ml / min;

[0179] Column temperature: 30 °C;

[0180] Injection volume: 10 μL;

[0181] Solvent: Acetonitrile: Water = 1:9 (V:V);

[0182] (2) Preparation of Sample Solution

[0183] The preparation of the sample solution and the injection detection method are the same as those in Example 1.

[0184] (3) Detect the blank solution, reference solution, and test solution according to the instruments and chromatographic conditions disclosed in step (1), record the chromatogram, and the chromatogram is as Figure 5 shown.

[0185] Comparative Example 12

[0186] According to the method and chromatographic conditions of chlorpheniramine maleate in the Chinese Pharmacopoeia, the specific detection steps are as follows:

[0187] (1) Detection conditions

[0188] Detection wavelength: 225 nm;

[0189] Chromatographic column: octadecylsilyl silica gel chromatographic column (Agilent ZORBAX SB C18), with a specification of 4.6 * 150 mm and a packing particle size of 5 μm;

[0190] Mobile phase A: phosphate buffer solution (take 11.5 g of ammonium dihydrogen phosphate, add an appropriate amount of water to dissolve it, add 1 ml of phosphoric acid, and dilute it to 1000 mL with water);

[0191] Mobile phase B: acetonitrile;

[0192] The gradient elution program is shown in Table 14:

[0193] Table 14 Gradient elution program

[0194] Time (min) Mobile Phase A / % Mobile Phase B / % 0 90 10 25 75 25 40 60 40 45 90 10 50 90 10

[0195] Flow rate: 1.2 mL / min;

[0196] Column temperature: room temperature;

[0197] Injection volume: 10 μL;

[0198] Solvent: acetonitrile: water = 1:9 (V:V);

[0199] (2) Preparation of sample solution

[0200] The preparation of the sample solution and the injection and detection methods are the same as those in Example 1.

[0201] (3) Detect the blank solution, reference solution, and test solution according to the instruments and chromatographic conditions disclosed in step (1), record the chromatogram, and the chromatogram is as Figure 6 shown.

[0202] From Figure 5 and Figure 6 , it can be seen that under the acidic system with phosphate buffer solution as the mobile phase, the retention capabilities of GTI-1 (2-aminopyridine-N-oxide), GTI-2 (2-bromopyridine-N-oxide), and GTI-3 (2-aminopyridine) are very weak, and the detection is greatly interfered by the solvent. Under such chromatographic conditions, the contents of GTI-1, GTI-2, and GTI-3 cannot be accurately quantified.

[0203] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate, characterized in that, Use a chromatographic column packed with octadecylsilyl silica gel, with ammonia aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution, and use high performance liquid chromatography to detect genotoxic impurities in chlorpheniramine maleate samples; the genotoxic impurities are 2-aminopyridine-N-oxide, 2-bromopyridine-N-oxide, and 2-aminopyridine.

2. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, The concentration of the ammonia aqueous solution is 1 mL / L.

3. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, The chromatographic column is a Welch Xtimate C18 chromatographic column, with a specification of 4.6 mm×150 mm, a packing particle size of 5 μm, and a column temperature of 30 °C.

4. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, wherein The program of the gradient elution is as follows: The above percentages are volume ratios.

5. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, The flow rate of the gradient elution is 1 mL / min.

6. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, In the high performance liquid chromatography, the injection volume is 5 μL.

7. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, In the high performance liquid chromatography, the detection wavelength is 225 nm.

8. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 1, characterized in that, The detection of genotoxic impurities in chlorpheniramine maleate samples includes the following steps: 1) Prepare a test solution and a reference solution containing genotoxic impurities; 2) Detect the test solution and the reference solution by high performance liquid chromatography, record the peak area of the corresponding genotoxic impurity in the test solution, denoted as A 供试品 , and the peak area of the genotoxic impurity in the reference solution, denoted as A 对照品 ; 3) Calculate the content ppm of genotoxic impurities in chlorpheniramine maleate according to the following formula; In the formula: W 对照品 The weighed quantity of the reference substance, mg; A 对照品 is the peak area of the genotoxic impurity in the reference solution; V 对照品 is the dilution factor of the reference substance; W 供试品 is the weighed sample amount of the test sample, mg; A 供试品 is the peak area of the genotoxic impurity in the test solution; V 供试品 is the dilution factor of the test sample.

9. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 8, characterized in that, The preparation of the reference solution containing genotoxic impurities includes the following steps: S1. Respectively take 23 mg of 2-aminopyridine-N-oxide, 2-bromopyridine-N-oxide, and 2-aminopyridine, and place them in 25 mL volumetric flasks respectively. Dissolve with acetonitrile and dilute to the scale, shake well, and prepare 2-aminopyridine-N-oxide stock solution, 2-bromopyridine-N-oxide stock solution, and 2-aminopyridine stock solution respectively; S2. Respectively transfer 1 mL of 2-aminopyridine-N-oxide stock solution, 2-bromopyridine-N-oxide stock solution, and 2-aminopyridine stock solution into a 25 mL volumetric flask, dilute to the scale with the solvent, and shake well to prepare a reference stock solution; S3. Transfer 1 mL of the reference stock solution into a 25 mL volumetric flask, dilute to the scale with the solvent, and shake well to prepare a reference solution.

10. The high performance liquid chromatography detection method for trace genotoxic impurities in chlorpheniramine maleate according to claim 9, characterized in that, The solvent is an acetonitrile aqueous solution, and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:9.

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